Basement membranes are thin layers of specialized extracellular matrix that provide supporting structure on which epithelial and endothelial cells grow, and that surround muscle or fat (Paulsson, M., 1992, Crit. Rev. Biochem. Mol. Biol. 27:93-127). Basement membranes are always associated with cells, and it has been well documented that basement membranes not only provide mechanical support, but also influence cellular behavior such as differentiation and proliferation. Vascular basement membranes are composed of macromolecules such as collagen, laminin, heparan sulfate proteoglycans, fibronectin and entactin (Timpl, R., 1996, Curr. Opin. Cell. Biol. 8:618-24). Functionally, collagen promotes cell adhesion, migration, differentiation and growth (Paulsson, M., 1992, Crit. Rev. Biochem. Mol. Biol. 27:93-127), and via these functions is presumed to play a crucial role in endothelial cell proliferation and behavior during angiogenesis, which is the process of formation of new blood vessels from pre-existing ones (Madri, J. A. et al., 1986, J. Histochem. Cytochem. 34:85-91; Folkman, J., 1972, Ann. Surg. 175:409-16). Angiogenesis is a complex process, and requires sprouting and migration of endothelial cells, proliferation of those cells, and their differentiation into tube-like structures and the production of a basement membrane matrix around the developing blood vessel. Additionally angiogenesis is a process critical for normal physiological events such as wound repair and endometrium remodeling (Folkman, J. et al., 1995, J. Biol. Chem. 267:10931-34). It is now well documented that angiogenesis is required for metastasis and growth of solid tumors beyond a few mm3 in size (Folkman, J., 1972, Ann. Surg. 175:409-16; Folkman, J., 1995, Nat. Med. 1:27-31). Expansion of tumor mass occurs not only by perfusion of blood through the tumor, but also by paracrine stimulation of tumor cells by several growth factors and matrix proteins produced by the new capillary endothelium (Folkman, J., 1995, Nat. Med. 1:27-31). Recently, a number of angiogenesis inhibitors have been identified, namely angiostatin (O'Reilly, M. S. et al., 1994, Cell 79:315-28), endostatin (O'Reilly, M. S. et al., 1997, Cell 88:277-85), restin (Ramchandran, R. et al., 1999, Biochem. Biophys. Res. Commun. 255:735-9) and pigment epithelilum-derived factor (PEDF) (Dawson, D. W. et al., 1999, Science 285:245-8).
Type IV collagen is expressed as six distinct α-chains, α1 through α6 (Prockop, D. J. et al., 1995, Annu. Rev. Biochem. 64:403-34), and assembled into triple helices. It further forms a network to provide a scaffold for other macromolecules in basement membranes. These α-chains are composed of three domains, the N-terminal 7S domain, the middle triple helical domain, and the C-terminal globular non-collagenous (NCl) domain (Timpl, R. et al., 1981, Eur. J. Biochem. 120:203-211). Several studies have shown that inhibitors of collagen metabolism have anti-angiogenic properties, supporting the notion that basement membrane collagen synthesis and deposition is crucial for blood vessel formation and survival (Maragoudakis, M. E. et al., 1994, Kidney Int. 43:147-50; Haralabopoulos, G. C. et al., 1994, Lab. Invest. 71:575-82). However, the precise role of collagen in basement membrane organization and angiogenesis is still not well understood.
Integrins are a family of important cell surface adhesion receptors which function as adhesive molecules for many compounds. They are involved in cell-cell or cell-extracellular matrix interactions, and both mediate cells' interactions with the extracellular matrix, and cause cells to bind with it. Integrins are αβ heterodimers, consisting of two non-covalently bound transmembrane glycoprotein subunits, the α subunit and the β subunit. All α subunits exhibit shared homology with each other, as do all of the β subunits. There are currently sixteen α subunits identified (α1 through α9, αD, αL, αM, αV, αX, αIIb and αIELb), and eight β subunits (β1 through β8), which form 22 different known combinations (β1 and α1 through α9; β1 and αV; β2 and αD, αL, αM and αX; β3 and αV and αX; β4 and α6; β5 and αV; β6 and αV; β7 and α4 and αIELb; β8 and αV). The pool of the available integrin subunits can be further increased by alternative splicing of the mRNA of some of the integrin subunits.
Integrins generally bind their ligands when the concentration of integrins at a particular spot on the cell surface is above a certain minimum threshold, forming a focal contact, or hemidesmosome. This combination of low binding affinity and formation of focal contacts enables integrins to bind both weakly and strongly, depending on the concentrations of integrin molecules.